1. Field of the Invention
The present invention relates to methods of preparing antihemophilic factor (AHF) from human plasma. AHF is now known to consist of several components, the component which is active in treating hemophilia A being Factor VIII:C.
2 Description of the Prior Art
Numerous patents and publications exist which relate to the preparation of AHF concentrates as part of the fractionation of human plasma. Such processes have been in commercial use for approximately 20 years, and numerous processing variations have been described, the vast majority of which are directed to the inherent problems in such processes, namely virus safety, yield, and specific activity of the resultant concentrate. Specific activity refers to the activity of the Factor VIII, expressed in international units, according to a currently accepted standard, per mg of total protein.
Although gel filtration or chromatography, except for affinity chromatography such as described in Zimmerman et al, Re. 32,011 (U.S. Pat. No. 4,361,509) is not, to the inventors' knowledge, in current commercial use, several chromatography processes have been described. It is important to note that all affinity chromatography or rDNA processes will result in AHF having detectable amounts of non-human protein.
For example, PCT Application Publication No. WO 86,04486 discloses a method for purifying AHF by "hydration additives", i.e. using column chromatography in the presence of sugars, polyols, amino acids or salts. The low yield of prior art chromatography processes is described.
The hydration additives serve to stabilize the AHF. Cryoprecipitate is dissolved in a buffer, aluminum hydroxide may be added and the supernatant collected. A PEG precipitation step is carried out. One or two column chromatography steps are then carried out, using resins such as QAE Sephadex A-25, QAE-Sepharose 4B or aminohexyl (A-H) Sepharose. The first chromatography step is based on anion exchange, the second on hydrophobic affinity.
Andersson, EP 197901, discloses a method for preparing fragments of AHF using immunoaffinity chromatography followed by HPLC on an anion-exchange adsorbent. The anion exchange adsorbent may be Mono Q gel or TSK DEAE 5 PW gel. Fragments are then obtained by incubation with thrombin.
Johnson, U.S. Pat. No. 4,397,841, discloses preparation of Factor VIII:C by fractionation of plasma with a sequence of adsorption steps employing polyelectrolyte copolymers in the presence of heparin. A suitable resin is a copolymer of ethylene and maleic anhydride.
Chavin et al, U.S. Pat. No. 4,495,175, disclose the preparation of highly purified AHF from AHF concentrate. The concentrate is subjected to a separation on the basis of Stokes' radius, which may be accomplished, for example, by gel permeation chromatography on cross-linked agarose (such as BioGel A-15M or Sepharose CL-4B). The pool is then concentrated by precipitation or diafiltration; calcium or magnesium cations are added to reduce the Stokes' radius, and a separation on the basis of Stokes' radius is again carried out.
Various other steps such as are employed in the present process have been disclosed in the prior art. However, as described below, novel and unexpected results and modifications are embraced by the present invention.
Liu et al, U.S. Pat. No. 4,170,639, for example disclose a process for preparing AHF comprising the steps of subjecting resolubilized cryoprecipitate to aluminum hydroxide adsorption at an acid pH and 4.degree. C.; filtration; and, optionally, ultrafiltration.
Rasmussen et al, U.S. Pat. No. 4,650,858, disclose a process for producing AHF using a 4% PEG precipitation step at 18-22.degree. C. to remove fibrinogen. This is followed by a second PEG precipitation step at 18-22.degree. C. with 12% PEG in the presence of an amino acid such as 2M glycine to precipitate the AHF.
Shanbrom, U.S. Pat. No. 4,069,216, discusses PEG precipitation as disclosed in the prior art, e.g. his U.S. Pat. No. 3,631,018 wherein room temperature precipitation necessitates a subsequent washing and/or glycine or alcohol precipitation step, since the PEG is used in high concentrations (10-12%). Cold precipitation using lower concentrations of PEG (21/2%) resulted in a less purified product.
Liautaud et al, U.S. Pat. No. 4,387,092, disclose an improvement to Shanbrom U.S. Pat. No. 4,069,216 in that the fibrinogen precipitation step is carried out at below 15.degree. C. with less than 4% polyol.
Polson, U.S. Pat. No. 3,415,804, discloses plasma fractionation with PEG at room temperature, around 20.degree. C. At 0-4% PEG, fibrinogen precipitated, gamma globulin precipitated at 4-8%, beta globulin at 8-12% and alpha-1 and alpha-2 globulins and albumins at greater than 12% PEG.
Finally, relevant prior art exists with regard to virus inactivation of AHF concentrates.
Neurath et al, U.S. Pat. No. 4,540,573, disclose viral inactivation of Factor VIII preparations through the use of tri-(n-butyl) phosphate (TNBP). It is there suggested that TNBP may be added to the plasma pool, and AHF can be separated from TNBP by a precipitation step, such as with glycine. In the Examples, TNBP is added to AHF solutions having 8-10 u/mL F.VIII activity.
Andersson et al, U.S. Pat. No. 4,168,300, disclose a method of removing hepatitis virus from plasma by adsorbing the HBsAg, or Av-antigen onto a beaded agarose gel, or a copolymer gel, having a hydrophobic ligand coupled thereto.
Lembach, U.S. Pat. No. 4,534,972, discloses the use of copper phenanthroline for viral inactivation of AHF preparations. The substance is added after fractionation and may be removed by diafiltration.